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A NOVEL PROCESS SAFEGUARDS GENOME INTEGRITY IN THE MAMMALIAN GERM LINE

保护哺乳动物生殖系基因组完整性的新工艺

基本信息

批准号：
10445720
负责人：
Piroska Edit Szabó
金额：
$ 51.9万
依托单位：
VAN ANDEL RESEARCH INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-06-20 至 2026-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10445720
关键词：
Adopted Affect Affinity B-DNA BTB/POZ Domain Binding Binding Sites Biochemical Biochemistry Biological Assay Biological Process Cell Culture Techniques Cells Cellular biology Chromosomal Rearrangement Chromosome Deletion Circular Dichroism DNA DNA Double Strand Break DNA Methylation DNA Modification Methylases DNA Sequence Rearrangement DNA Structure DNA-Binding Proteins Dependence Development EMSA Enzymes Epigenetic Process Generations Genetic Genetic Models Genome Germ Cells Germ Lines Germ-Line Mutation Grant Hand Human Immunohistochemistry In Vitro Left Location Mammalian Cell Maps Measures Methods Methylation Methyltransferase Molecular Monitor Mus Mutant Strains Mice Nucleosomes Organism Population Pregnancy Process Proteins Purines Pyrimidine Refractory Role Short Tandem Repeat Site Spermatocytes Tandem Repeat Sequences Testing Time Transgenic Mice Variant Z-Form DNA Zinc Fingers base epigenome epigenomics fetal genome integrity genome-wide in vivo male mutant mutation assay next generation novel sperm cell

项目摘要

PROJECT SUMMARY Simple purine–pyrimidine (Pu/Py) repeats (PPRs) are known to form the left-handed, fragile Z-DNA structure. Such repeats are also known to be highly mutagenic, inducing large chromosomal deletions and rearrangements in the cells of higher organisms. The mutagenic effects of Z-DNA would be the most detrimental to a species if breaks occurred uncontrollably in the germ line and mutations occurring at unrepaired breaks were passed on to the next generation. We discovered a novel biological process in the mammalian germ line that controls Z- DNA structure at PPRs. Using the Zbtb43 mutant mouse genetic model and our transgenic mouse line that allows us to isolate germ cells, we found that a previously uncharacterized DNA binding protein, ZBTB43 remodels Z-DNA structure and protects from double-strand breaks in fetal male germ cells in vivo. By biochemical assays we found that ZBTB43 binds to PPR-rich DNA sites in the genome in vitro. ZBTB43 binding sites form Z-DNA and cause large genomic rearrangements in mammalian cells. By in vivo epigenome mapping we detected Z-DNA in mutant germ cells at the locations where ZBTB43 binding occurs in wild-type prospermatogonia. We hypothesize, therefore, that ZBTB43 safeguards genome integrity in the germ line by binding and eliminating Z-DNA at PPRs. In addition, we found that by eliminating Z-DNA, ZBTB43 promotes de novo methylation at PPRs during the time of global epigenetic remodeling. We propose to pursue the following Aims, using a combination of genetic, cell biology, biochemistry, and epigenomic approaches. In Aim 1, we will test the working hypothesis that ZBTB43 eliminates Z-DNA structure in vivo by directly binding to PPRs in fetal male germ cells. We will determine 1) the spatial and temporal changes of the Z-DNA structure in fetal male germ cells in the presence or absence of ZBTB43 protein in vivo; 2) the dependence of the Z-DNA remodeling process on the direct binding of ZBTB43 to the Z-DNA structure in vivo; and 3) the molecular requirements of ZBTB43 action on Z-DNA. In Aim 2, we will test the working hypothesis that ZBTB43 facilitates de novo DNA methylation in prospermatogonia indirectly by eliminating Z-DNA, thus revealing the sequences as substrates for de novo methyltransferases. We will map DNA methylation in the presence and absence of ZBTB43 during the epigenome remodeling process in fetal male germ cells. We will test whether de novo DNMTs methylate Z- DNA substrates in vitro. We will test whether ZBTB43 affects nucleosome occupancy at PPRs. In Aim 3, we will test the hypothesis that Z-DNA is mutagenic in the germ line and that ZBTB43 has evolved to manage that burden. We will map double-strand breaks in mutant fetal germ cells, test the anti-mutagenic effect of ZBTB43 in cell culture, and search for genomic rearrangements in sperm of Zbtb43 mutant males. By the end of the grant period, we will have identified and characterized the first example of how a DNA binding protein protects genome integrity and targets de novo DNA methylation by controlling Z-DNA structure in the mammalian germ line.

项目总结